Psilocybe Cubensis Research Articles

Association of Bacterial Communities with Psychedelic Mushroom and Soil as Revealed in 16S rRNA Gene Sequencing.

Microbial communities' resident in the mushroom fruiting body and the soil around it play critical roles in the growth and propagation of the mushroom. Among the microbial communities associated with psychedelic mushrooms and the rhizosphere soil, bacterial communities are considered vital since their presence greatly influences the health of the mushrooms. The present study aimed at finding the microbiota present in the psychedelic mushroom Psilocybe cubensis and the soil the mushroom inhabits. The study was conducted at two different locations in Kodaikanal, Tamil Nadu, India. The composition and structure of microbial communities in the mushroom fruiting body and the soil were deciphered. The genomes of the microbial communities were directly assessed. High-throughput amplicon sequencing revealed distinct microbial diversity in the mushroom and the related soil. The interaction of environmental and anthropogenic factors appeared to have a significant impact on the mushroom and soil microbiome. The most abundant bacterial genera were Ochrobactrum, Stenotrophomonas, Achromobacter, and Brevundimonas. Thus, the study advances the knowledge of the composition of the microbiome and microbial ecology of a psychedelic mushroom, and paves the way for in-depth investigation of the influence of microbiota on the mushroom, with special emphasis on the impact of bacterial communities on mushroom growth. Further studies are required for a deeper understanding of the microbial communities that influence the growth of P. cubensis mushroom.

In silico characterization of the psilocybin biosynthesis pathway

Nearly all mushrooms of the Psilocybe genus contain the natural product psilocybin, which is a psychoactive alkaloid derived from l-tryptophan. Considering their use in ancient times, as well as their psychedelic properties, these mushrooms have re-emerged with psychotherapeutic potential for treating depression, which has triggered increased pharmaceutical interest. However, the psilocybin biosynthesis pathway was only recently defined and, as such, little exists in the way of structural data. Accordingly, the aim of this study was to structurally characterize this pathway by generating homology models for the four Psilocybe cubensis enzymes involved in psilocybin biosynthesis (PsiD, a decarboxylase; PsiH, a monooxygenase; PsiK, a phosphotransferase; PsiM, a methyltransferase). Following initial model generation and alignment with the identified structural templates, repeated refinement of the models was carried out using secondary structure prediction, geometry evaluation, energy minimization, and molecular dynamics simulations in water. The final models were then evaluated using molecular docking interactions with their substrates, i.e., psilocybin precursors (l-tryptophan, tryptamine, 4-hydroxytryptamine, and norbaeocystin/baeocystin), all of which generated feasible binding modes for the expected biotransformation. Further plausibility of the psilocybin → aeruginascin, 4-hydroxytryptamine → norpsilocin, and tryptamine → N,N-dimethyltryptamine conversions, all mediated by the generated model for PsiM, suggests valid routes of formation for these key secondary metabolites. The structural characterization of these enzymes and their binding modes which emerged from this study can lead to a better understanding of psilocybin synthesis, thereby paving the way for the development of novel substrates and selective inhibitors, as well as improved biotechnological manipulation and production of psilocybin in vitro.

DNA Authentication and Chemical Analysis of Psilocybe Mushrooms Reveal Widespread Misdeterminations in Fungaria and Inconsistencies in Metabolites.

The mushroom genus Psilocybe is best known as the core group of psychoactive mushrooms, yet basic information on their diversity, taxonomy, chemistry, and general biology is still largely lacking. In this study, we reexamined 94 Psilocybe fungarium specimens, representing 18 species, by DNA barcoding, evaluated the stability of psilocybin, psilocin, and their related tryptamine alkaloids in 25 specimens across the most commonly vouchered species (Psilocybe cubensis, Psilocybe cyanescens, and Psilocybe semilanceata), and explored the metabolome of cultivated P. cubensis. Our data show that, apart from a few well-known species, the taxonomic accuracy of specimen determinations is largely unreliable, even at the genus level. A substantial quantity of poor-quality and mislabeled sequence data in public repositories, as well as a paucity of sequences derived from types, further exacerbates the problem. Our data also support taxon- and time-dependent decay of psilocybin and psilocin, with some specimens having no detectable quantities of them. We also show that the P. cubensis metabolome possibly contains thousands of uncharacterized compounds, at least some of which may be bioactive. Taken together, our study undermines commonly held assumptions about the accuracy of names and presence of controlled substances in fungarium specimens identified as Psilocybe spp. and reveals that our understanding of the chemical diversity of these mushrooms is largely incomplete. These results have broader implications for regulatory policies pertaining to the storage and sharing of fungarium specimens as well as the use of psychoactive mushrooms for recreation and therapy. IMPORTANCE The therapeutic use of psilocybin, the active ingredient in "magic mushrooms," is revolutionizing mental health care for a number of conditions, including depression, posttraumatic stress disorder (PTSD), and end-of-life care. This has spotlighted the current state of knowledge of psilocybin, including the organisms that endogenously produce it. However, because of international regulation of psilocybin as a controlled substance (often included on the same list as cocaine and heroin), basic research has lagged far behind. Our study highlights how the poor state of knowledge of even the most fundamental scientific information can impact the use of psilocybin-containing mushrooms for recreational or therapeutic applications and undermines critical assumptions that underpin their regulation by legal authorities. Our study shows that currently available chemical studies are mainly inaccurate, irreproducible, and inconsistent, that there exists a high rate of misidentification in museum collections and public databases rendering even names unreliable, and that the concentration of psilocybin and its tryptamine derivatives in three of the most commonly collected Psilocybe species (P. cubensis, P. cyanescens, and P. semilanceata) is highly variable and unstable in museum specimens spanning multiple decades, and our study generates the first-ever insight into the highly complex and largely uncharacterized metabolomic profile for the most commonly cultivated magic mushroom, P. cubensis.

Characterization of the Gateway Decarboxylase for Psilocybin Biosynthesis.

The l-tryptophan decarboxylase PsiD catalyzes the initial step of the metabolic cascade to psilocybin, the major indoleethylamine natural product of the "magic" mushrooms and a candidate drug against major depressive disorder. Unlike numerous pyridoxal phosphate (PLP)-dependent decarboxylases for natural product biosyntheses, PsiD is PLP-independent and resembles type II phosphatidylserine decarboxylases. Here, we report on the in vitro biochemical characterization of Psilocybe cubensis PsiD along with in silico modeling of the PsiD structure. A non-canonical serine protease triad for autocatalytic cleavage of the pro-protein was predicted and experimentally verified by site-directed mutagenesis.

Microdosing with psilocybin mushrooms: a double-blind placebo-controlled study

The use of low sub-perceptual doses of psychedelics (“microdosing”) has gained popularity in recent years. Although anecdotal reports claim multiple benefits associated with this practice, the lack of placebo-controlled studies severely limits our knowledge of microdosing and its effects. Moreover, research conducted in standard laboratory settings could fail to capture the motivation of individuals engaged or planning to engage in microdosing protocols, thus underestimating the likelihood of positive effects on creativity and cognitive function. We recruited 34 individuals starting to microdose with psilocybin mushrooms (Psilocybe cubensis), one of the materials most frequently used for this purpose. Following a double-blind placebo-controlled experimental design, we investigated the acute and short-term effects of 0.5 g of dried mushrooms on subjective experience, behavior, creativity (divergent and convergent thinking), perception, cognition, and brain activity. The reported acute effects were significantly more intense for the active dose compared to the placebo, but only for participants who correctly identified their experimental condition. These changes were accompanied by reduced EEG power in the theta band, together with preserved levels of Lempel-Ziv broadband signal complexity. For all other measurements there was no effect of microdosing except for few small changes towards cognitive impairment. According to our findings, low doses of psilocybin mushrooms can result in noticeable subjective effects and altered EEG rhythms, but without evidence to support enhanced well-being, creativity and cognitive function. We conclude that expectation underlies at least some of the anecdotal benefits attributed to microdosing with psilocybin mushrooms.

Nephrotoxic Mushroom Poisoning: Global Epidemiology, Clinical Manifestations, and Management.

Because mushroom poisonings are increasing worldwide after ingestions of known, newly described, and formerly considered edible species, the objectives of this review are to describe the global epidemiology of nephrotoxic mushroom poisonings, to identify nephrotoxic mushrooms, to present a toxidromic approach to earlier diagnoses of nephrotoxic mushroom poisonings based on the onset of acute renal failure, and to compare the outcomes of renal replacement management strategies. Internet search engines were queried with the keywords to identify scientific articles on nephrotoxic mushroom poisonings and their management during the period of 1957 to the present. Although hepatotoxic, amatoxin-containing mushrooms cause most mushroom poisonings and fatalities, nephrotoxic mushrooms, most commonly Cortinarius species, can cause acute renal insufficiency and failure. Several new species of nephrotoxic mushrooms have been identified, including Amanita proxima and Tricholoma equestre in Europe and Amanita smithiana in the United States and Canada. In addition, the edible, hallucinogenic mushroom Psilocybe cubensis has been noted recently via mass spectrometry as a rare cause of acute renal insufficiency. Renal replacement therapies including hemodialysis are often indicated in the management of nephrotoxic mushroom poisonings, with renal transplantation reserved for extracorporeal treatment failures.

Anti-Inflammatory Effects of Four Psilocybin-Containing Magic Mushroom Water Extracts in vitro on 15-Lipoxygenase Activity and on Lipopolysaccharide-Induced Cyclooxygenase-2 and Inflammatory Cytokines in Human U937 Macrophage Cells.

PurposeDuring a pathological inflammation, macrophages are activated to produce accumulation of inflammatory mediators such as induced-cyclooxygenase-2 (COX-2), 15-lipoxygenase (15-LOX) and pro-inflammatory cytokines. Pathological inflammation is a significant problem in many chronic diseases. As a result, more research into natural remedies with anti-inflammatory potential is crucial. Since ancient times, psilocybin-containing mushrooms, also known as magic mushrooms, were used for mind healing and also to advance the quality of life. However, not much is known about their anti-inflammatory potential. This study aimed at investigating the anti-inflammatory effects of four psilocybin-containing mushrooms (Panaeolus cyanescens, Psilocybe natalensis, Psilocybe cubensis and Psilocybe cubensis leucistic A+ strain) from genus Panaeolus and Psilocybe for the first time in vitro on 15-LOX activity and also on lipopolysaccharide (LPS)-induced inflammation in human U937 macrophage cells.MethodsMushrooms were grown and extracted with boiling hot water. Effects of the four water extracts on 15-LOX activity were determined. Confluent human U937 cells were differentiated with phorbol 12-myristate 13-acetate and treated with the hot-water extracts (25 and 50 µg/mL) 2 hours before being stimulated with 1 µg/mL LPS over 24 hours. Quercetin was used as a positive control. Control cells were differentiated but not LPS-induced nor treated. Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-10 concentrations were measured. Levels of COX-2 and mitochondrial activity were also determined.ResultsThe four water extracts had poor 15-LOX inhibition activity with IC50 > 250 µg/mL. Extracts were safe at the concentration studied and inhibited the LPS-induced production of pro-inflammatory mediators, TNF-α and IL-1β significantly and lowered IL-6 and COX-2 concentrations in treated human U937 macrophage cells. Water extracts also increased percentage viability of treated cells and levels of anti-inflammatory IL-10 non-significantly.ConclusionThe study suggested that the hot-water extracts of the four psilocybin-containing magic mushrooms have potential anti-inflammatory effects executed by down-regulating pro-inflammatory mediators.

A draft reference assembly of the Psilocybe cubensis genome

We describe the use of high-fidelity single molecule sequencing to assemble the genome of the psychoactive Psilocybe cubensis mushroom. The genome is 46.6Mb, 46% GC, and in 32 contigs with an N50 of 3.3Mb. The BUSCO completeness scores are 97.6% with 1.2% duplicates. The Psilocybin synthesis cluster exists in a single 3.2Mb contig. The dataset is available from NCBI BioProject with accessions PRJNA687911 and PRJNA700437.

A draft sequence reference of the Psilocybe cubensis genome.

We describe the use of high-fidelity single molecule sequencing to assemble the genome of the psychoactive Psilocybe cubensis mushroom. The genome is 46.6Mb, 46% GC, and in 32 contigs with an N50 of 3.3Mb. The BUSCO completeness scores are 97.6% with 1.2% duplicates. The Psilocybin synthesis cluster exists in a single 3.2Mb contig. The dataset is available from NCBI BioProject with accessions PRJNA687911 and PRJNA700437.

Development of a physiologically based pharmacokinetic (PBPK) model of psilocybin and psilocin from magic mushroom in rats and humans

Background: Psilocybin (PB) is a psychoactive compound commonly found in magic mushroom (Psilocybe cubensis). PB is quickly converted by the body to psilocin (PI), which has a psychedelic effect through the activation of the 5-HT2A receptor in the brain. The objective of this study is to develop a physiologically based pharmacokinetic (PBPK) model of PB and PI in rats and humans for predicting concentrations of the psychoactive substance in the brain. Methods: Following a search in PubMed, three studies were retrieved and information concerning concentration-time profiles of PI were extracted from the selected studies. In the study in rats, PI was orally administered with a dose of 10.1 mg/kg. There were two studies in humans following a single intravenous dose of PB (1 mg) and oral dose of PB (0.224 mg/kg and 0.3 mg/kg). Berkeley Madonna software was used for computer coding and simulations. The developed PBPK model consisted of seven organ compartments (i.e. lung, heart, brain, fat, muscle, kidney, and liver). Results: The simulations show a good agreement between observed and simulated data, although results for oral administration in rats and humans showed under-predictions and results for intravenous administration in humans showed over-predictions. Conclusions: A PBPK model of PB and PI in rats and humans was developed and could predict concentration-time profiles of PI in plasma, particularly in the brain, following intravenous and oral administration of PB. This model may be useful for a safer dosage regimen of PB for patients with some disorders.

Chemoenzymatic Synthesis of 5-Methylpsilocybin: A Tryptamine with Potential Psychedelic Activity.

A novel analogue of psilocybin was produced by hybrid chemoenzymatic synthesis in sufficient quantity to enable bioassay. Utilizing purified 4-hydroxytryptamine kinase from Psilocybe cubensis, chemically synthesized 5-methylpsilocin (2) was enzymatically phosphorylated to provide 5-methylpsilocybin (1). The zwitterionic product was isolated from the enzymatic step with high purity utilizing a solvent-antisolvent precipitation approach. Subsequently, 1 was tested for psychedelic-like activity using the mouse head-twitch response assay, which indicated activity that was more potent than the psychedelic dimethyltryptamine, but less potent than that of psilocybin.

A Forensic Detection Method for Hallucinogenic Mushrooms via High-Resolution Melting (HRM) Analysis.

In recent years, trafficking and abuse of hallucinogenic mushrooms have become a serious social problem. It is therefore imperative to identify hallucinogenic mushrooms of the genus Psilocybe for national drug control legislation. An internal transcribed spacer (ITS) is a DNA barcoding tool utilized for species identification. Many methods have been used to discriminate the ITS region, but they are often limited by having a low resolution. In this study, we sought to analyze the ITS and its fragments, ITS1 and ITS2, by using high-resolution melting (HRM) analysis, which is a rapid and sensitive method for evaluating sequence variation within PCR amplicons. The ITS HRM assay was tested for specificity, reproducibility, sensitivity, and the capacity to analyze mixture samples. It was shown that the melting temperatures of the ITS, ITS1, and ITS2 of Psilocybe cubensis were 83.72 ± 0.01, 80.98 ± 0.06, and 83.46 ± 0.08 °C, and for other species, we also obtained species-specific results. Finally, we performed ITS sequencing to validate the presumptive taxonomic identity of our samples, and the sequencing output significantly supported our HRM data. Taken together, these results indicate that the HRM method can quickly distinguish the DNA barcoding of Psilocybe cubensis and other fungi, which can be utilized for drug trafficking cases and forensic science.

Multi-locus identification of Psilocybe cubensis by high-resolution melting (HRM)

Hallucinogenic mushroom is a kind of toxic strain containing psychoactive tryptamine substances such as psilocybin, psilocin and ibotenic acid, etc. The mushrooms containing hallucinogenic components are various, widely distributed and lack of standard to define, which made a great challenge to identification. Traditional identification methods, such as morphology and toxicology analysis, showed shortcomings in old or processed samples, while the DNA-based identification of hallucinogenic mushrooms would allow to identify these samples due to the stability of DNA. In this paper, four primer sets are designed to target Psilocybe cubensis DNA for increasing resolution of present identification method, and the target markers include largest subunit of RNA polymerase II (marked as PC-R1), psilocybin-related phosphotransferase gene (marked as PC-PT), glyceraldehyde 3-phosphate dehydrogenase (marked as PC-3) and translation EF1α (marked as PC-EF). Real-time PCR with high-resolution melting (HRM) assay were used for the differentiation of the fragments amplified by these primer sets, which were tested for specificity, reproducibility, sensitivity, mixture analysis and multiplex PCR. It was shown that the melting temperatures of PC-R1, PC-PT, PC-3 and PC-EF of P. cubensis were (87.93 ± 0.12) °C, (82.21 ± 0.14) °C, (79.72 ± 0.12) °C and (80.11 ± 0.19) °C in our kinds of independent experiments. Significant HRM characteristic can be shown with a low concentration of 62.5 pg/µL DNA sample, and P. cubensis could be detected in mixtures with Homo sapiens or Cannabis sativa. In summary, the method of HRM analysis can quickly and specifically distinguish P. cubensis from other species, which could be utilized for forensic science, medical diagnosis and drug trafficking cases. Supplemental data for this article are available online at https://doi.org/10.1080/20961790.2021.1875580.

Effects and safety of Psilocybe cubensis and Panaeolus cyanescens magic mushroom extracts on endothelin-1-induced hypertrophy and cell injury in cardiomyocytes

Prevalence of major depression in people with chronic heart failure is higher than in normal populations. Depression in heart failure has become a major issue. Psilocybin-containing mushrooms commonly known as magic mushrooms, have been used since ancient times for their mind healing properties. Their safety in cardiovascular disease conditions is not fully known and may pose as a risk for users suffering from these illnesses. Study investigates the effects and safety of Psilocybe cubensis and Panaeolus cyanescens magic mushrooms use from genus Psilocybe and Panaeolus respectively, in a pathological hypertrophy conditions in which endothelin-1 disorder is a contributor to pathogenesis. We examined the effects of the mushrooms extracts on endothelin-1-induced hypertrophy and tumor necrosis factor-α (TNF- α)-induced cell injury in H9C2 cardiomyocytes. Mushrooms were oven dried and extracted with cold and boiling-hot water. H9C2 cardiomyocytes were induced with endothelin-1 prior to treatment with extracts over 48 h. Cell injury was stimulated with TNF-α. Results proposed that the water extracts of Panaeolus cyanescens and Psilocybe cubensis did not aggravate the pathological hypertrophy induced by endothelin-1 and also protected against the TNF-α-induced injury and cell death in concentrations used. Results support medicinal safe use of mushrooms under controlled conditions and cautioned use of higher concentrations.

Stability of psilocybin and its four analogs in the biomass of the psychotropic mushroom Psilocybe cubensis.

Psilocybin, psilocin, baeocystin, norbaeocystin, and aeruginascin are tryptamines structurally similar to the neurotransmitter serotonin. Psilocybin and its pharmacologically active metabolite psilocin in particular are known for their psychoactive effects. These substances typically occur in most species of the genus Psilocybe (Fungi, Strophariaceae). Even the sclerotia of some of these fungi known as "magic truffles" are of growing interest in microdosing due to them improving cognitive function studies. In addition to microdosing studies, psilocybin has also been applied in clinical studies, but only its pure form has been administrated so far. Moreover, the determination of tryptamine alkaloids is used in forensic analysis. In this study, freshly cultivated fruit bodies of Psilocybe cubensis were used for monitoring stability (including storage and processing conditions of fruiting bodies). Furthermore, mycelium and the individual parts of the fruiting bodies (caps, stipes, and basidiospores) were also examined. The concentration of tryptamines in final extracts was analyzed using ultra-high-performance liquid chromatography coupled with mass spectrometry. No tryptamines were detected in the basidiospores, and only psilocin was present at 0.47 wt.% in the mycelium. The stipes contained approximately half the amount of tryptamine alkaloids (0.52 wt.%) than the caps (1.03 wt.%); however, these results were not statistically significant, as the concentration of tryptamines in individual fruiting bodies is highly variable. The storage conditions showed that the highest degradation of tryptamines was seen in fresh mushrooms stored at -80°C, and the lowest decay was seen in dried biomass stored in the dark at room temperature.

Immunochemical monitoring of psilocybin and psilocin to identify hallucinogenic mushrooms

Development of rapid and reliable immunochemical methods for monitoring psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine; Pyb) and psilocin (dephosphorylated metabolite; Psi), the psychoactive compounds contained within hallucinogenic mushrooms (magic mushrooms), is desirable in order to identify these mushrooms and regulate their illicit use. Because no antibody was publicly available for this purpose, we generated two independent monoclonal antibodies (mAbs) against Pyb or Psi, and then developed enzyme-linked immunosorbent assays (ELISAs) by using them. To generate the specific antibodies, novel immunogenic conjugates were prepared by linking Pyb or Psi molecules to carrier proteins by modifying their 2-(N,N-dimethylamino)ethyl side chains. Spleen cells from mice immunized with these conjugates were fused with P3/NS1/1-Ag4-1 myeloma cells, and hybridoma clones secreting anti-Pyb and anti-Psi mAbs were established. These mAbs were characterized for their biochemical features and then applied to competitive ELISAs, which used microplates coated with Pyb or Psi linked with albumin. These ELISAs enabled the determination of Pyb or Psi with measurable ranges of ca. 0.20−20 or 0.040−2.0 μg/assay (limit of detection was 0.14 or 0.029 μg/assay), respectively. The related tryptamines were satisfactorily discriminated as exemplified by the cross-reactivity of the ELISA to determine Pyb (or Psi) with Psi (or Pyb) that were found to be 2.8 % (or <0.5 %), respectively. The Pyb and Psi contents in a dried powder of the hallucinogenic mushroom, Psilocybe cubensis, were determined to be 0.39 and 0.32 (w/w)%, respectively. The ELISAs developed using the current mAbs are promising tools for identifying illegal hallucinogenic mushrooms.

Metabolic engineering of Saccharomyces cerevisiae for the de novo production of psilocybin and related tryptamine derivatives

Psilocybin is a tryptamine-derived psychoactive alkaloid found mainly in the fungal genus Psilocybe, among others, and is the active ingredient in so-called “magic mushrooms”. Although its notoriety originates from its psychotropic properties and popular use as a recreational drug, clinical trials have recently recognized psilocybin as a promising candidate for the treatment of various psychological and neurological afflictions. In this work, we demonstrate the de novo biosynthetic production of psilocybin and related tryptamine derivatives in Saccharomyces cerevisiae by expression of a heterologous biosynthesis pathway sourced from Psilocybe cubensis. Additionally, we achieve improved product titers by supplementing the pathway with a novel cytochrome P450 reductase from P. cubensis. Further rational engineering resulted in a final production strain producing 627 ± 140 mg/L of psilocybin and 580 ± 276 mg/L of the dephosphorylated degradation product psilocin in triplicate controlled fed-batch fermentations in minimal synthetic media. Pathway intermediates baeocystin, nor norbaeocystin as well the dephosphorylated baeocystin degradation product norpsilocin were also detected in strains engineered for psilocybin production. We also demonstrate the biosynthetic production of natural tryptamine derivative aeruginascin as well as the production of a new-to-nature tryptamine derivative N-acetyl-4-hydroxytryptamine. These results lay the foundation for the biotechnological production of psilocybin in a controlled environment for pharmaceutical applications, and provide a starting point for the biosynthetic production of other tryptamine derivatives of therapeutic relevance.

Taxonomic identification and bioactive compounds characterization of Psilocybe cubensis DPT1 to probe its antibacterial and mosquito larvicidal competency

Mushrooms have an important role in sustainability since they have long been used as valuable food source and traditional medicine around the world. Regrettably, they are among the most rigorously affected populations, along with several plants and animals, due to the destructive activities of mankind. Thus the authentication and conservation of mushroom species are constantly needed to exploit the remarkable potential in them. In this perspective, an attempt has been made to identify and assess the biological attributes of psychedelic mushrooms collected from Kodaikanal, Tamil Nadu, India. The macromorphological features of the psychedelic mushroom DPT1 helped its presumptive identification and the molecular characters depicted by DNA marker revealed its close relationship with the genus Psilocybe. Accordingly, the psychedelic mushroom was identified as Psilocybe cubensis DPT1 and its crude ethyl acetate extract on analysis revealed the occurrence of phytoconstituents like alkaloids, flavonoids, tannins, saponins and carbohydrates. Moreover, it exhibited 80% larvicidal activity against Culex quinquefasciatus mosquito at 800 ppm concentration and an array of antibacterial effects with utmost susceptibility of Proteus vulgaris, and the identification of bioactive compounds by different analytical techniques substantiate that the bioactivities might be due to the presence of phytochemicals. The results of the study indicated that the extract of P. cubensis DPT1 having notable antibacterial and mosquito larvicidal efficacies which could be probed further for the isolation of medicinally important as well as bio-control compounds.

S-Adenosyl-l-Methionine Salvage Impacts Psilocybin Formation in "Magic" Mushrooms.

Psychotropic Psilocybe mushrooms biosynthesize their principal natural product psilocybin in five steps, among them a phosphotransfer and two methyltransfer reactions, which consume one equivalent of 5′‐adenosine triphosphate (ATP) and two equivalents of S‐adenosyl‐l‐methionine (SAM). This short but co‐substrate‐intensive pathway requires nucleoside cofactor salvage to maintain high psilocybin production rates. We characterized the adenosine kinase (AdoK) and S‐adenosyl‐l‐homocysteine (SAH) hydrolase (SahH) of Psilocybe cubensis. Both enzymes are directly or indirectly involved in regenerating SAM. qRT‐PCR expression analysis revealed an induced expression of the genes in the fungal primordia and carpophores. A one‐pot in vitro reaction with the N‐methyltransferase PsiM of the psilocybin pathway demonstrates a concerted action with SahH to facilitate biosynthesis by removal of accumulating SAH.

Injury-Triggered Blueing Reactions of Psilocybe "Magic" Mushrooms.

Upon injury, psychotropic psilocybin‐producing mushrooms instantly develop an intense blue color, the chemical basis and mode of formation of which has remained elusive. We report two enzymes from Psilocybe cubensis that carry out a two‐step cascade to prepare psilocybin for oxidative oligomerization that leads to blue products. The phosphatase PsiP removes the 4‐O‐phosphate group to yield psilocin, while PsiL oxidizes its 4‐hydroxy group. The PsiL reaction was monitored by in situ 13C NMR spectroscopy, which indicated that oxidative coupling of psilocyl residues occurs primarily via C‐5. MS and IR spectroscopy indicated the formation of a heterogeneous mixture of preferentially psilocyl 3‐ to 13‐mers and suggest multiple oligomerization routes, depending on oxidative power and substrate concentration. The results also imply that phosphate ester of psilocybin serves a reversible protective function.